The present invention is related to the crystallization of cellulase enzymes. More particularly, the present invention relates to selective crystallization using salt of cellulase enzymes in an aqueous solution produced by, for example, the fermentation of microbial organisms such as filamentous fungi, yeast or bacteria.
Intensive research efforts have been directed to the precipitation and crystallization of enzymes as a means of purification and preparation of enzyme products. For example, in U.S. Pat. No. 4,659,667, a process is disclosed for the recovery of an enzyme from solution by concentrating to supersaturation the enzyme-containing solution at pH near the isoelectric point of the enzyme, inducing crystallization and recovering the crystallized final product. Inducing crystallization is achieved by allowing the enzymes to spontaneously crystallize upon concentration or by seeding, sound, stirring or scratching the inner surface of the container. Crystallization of alpha-amylase is exemplified.
In PCT Publication No. WO 89/08703, a process is described for the crystallization of subtilisin by adding a halide salt, such as sodium chloride or calcium chloride, to a concentrated subtilisin solution of at least about 40 grams per liter at temperatures less than 10.degree. C.
In PCT Publication No. WO 91/09943, a method for the crystallization of enzymes is disclosed which is characterized by using as a starting material an aqueous solution containing liquid with a relatively high enzyme purity and a concentration of enzyme of about at least 5 grams per liter and adding as a crystallization agent an easily soluble salt of the non-halide type to a concentration which is considerably smaller than the amount necessary to precipitate the enzymes in an amorphous form. Crystallization of certain subtilisin enzymes is exemplified.
In EP 549,048, a method for the preparation of highly purified alkaline protease from Bacillus licheniformis or Bacillus alcalophilus is disclosed which is characterized by adding hydrolytic enzymes and sodium chloride to concentrated alkaline protease, incubating the mixture at a temperature above 20.degree. C. to separate the alkaline protease from hydrolyzed polymeric impurities and collecting the purified precipitate. However, the precipitate was predominantly amorphous.
Methods for preparing cellulase crystals for the purpose of x-ray crystallography have been successful on a small scale. For example, Bergfors et al., J. Mol. Biol., vol. 209, no. 1, pp. 167-169 (1989) illustrate crystallization using the hanging drop method of the core protein of cellobiohydrolase II from Trichoderma reesei and subsequent study of the crystals to determine tertiary structure. In Wilson, Crit. Rev. Biochem., vol. 12(1/2), pp. 45-63 (1992) the 30 K catalytic subunit of the E2 cellulase from T. fusca was crystallized using ammonium sulfate as a precipitant to determine the tertiary structure. Importantly, Wilson points out that the numerous attempts to crystallize the T. fusca cellulases E2, E3 and E5 in the prior art using the hanging drop method with both ammonium sulfate and polyethylene glycol had failed. Thus, although crystallization of certain enzymes has been successful according to the means described above, the crystallization of cellulases has remained problematic with no known method for such crystallization on a large scale basis.
In spite of these advances in the field of enzyme crystallization in general, and occurrences of crystallization of cellulases, reported in the scientific literature, for the purpose of crystallographic studies, inexpensive and efficient crystallization of cellulase enzymes suitable for large scale production has remained problematic in industry. In fact, no commercially feasible process has been described for the crystallization of cellulases in terms of producing a low cost, high yield, rapid and highly purified cellulase enzyme in a simple manner.
To the contrary, Applicants have discovered that the employment of a specific group of salts and a specific temperature range provides the capability of selectively purifying through crystallization a commercially important subgroup of cellulases (i.e., those which lack a distinct cellulose binding domain) over other cellulases which possess a cellulose binding domain.
Further, any protein can be precipitated with enough salt, however, the industry prefers crystalline enzymes for further processing into, i.e., granules or immobilized enzymes. The present invention provides methods of obtaining a cellulase enzyme product.